Protein sequencing has traditionally been carried out using Edman degradation. However, the technique is slow and can require significant amounts of the protein. In order to overcome the disadvantages of Edman degradation, several new techniques have been developed. One technique is based on the separation of complex protein samples by two-dimensional gel electrophoresis and the subsequent sequential identification of the separated protein species (Ducret et al. (1998) Protein Sci. 7: 706-719). In a further refinement, the separated proteins are analyzed by mass spectrometric techniques and the protein and peptide mass spectral data correlated with sequence databases, thereby allowing for the rapid identification of proteins and peptides. However, the technique is sequential in nature, thereby limiting the number of samples that can be processed. Moreover, the method is difficult to automate.
In another method, called Isotope Coded Affinity Tags (ICAT), the protein-containing sample is derivatized with a cysteine specific reagent which contains a heavy/light form. After tagging, the samples are pooled, digested and the tagged fragments separated by affinity chromatography. The isolated fragments are subsequently analyzed by LC-MS or MALDI-TOFMS (WO 00/11208 to Aebersold et al.). The method thus assumes that every peptide or protein contains a cysteine that can be derivatized and that protein identity can be ascertained from the thiol-containing peptide fragment. In a related technique, termed Phosphoprotein Isotope Coded Affinity Tag (PhIAT), the O-phosphorylation sites of serine, threonine and tyrosine are isotopically enriched, the proteins separated and analyzed by mass spectroscopy (Goshe et al. (2001) Anal. Chem. 73: 2578-2586). The PhIAT methodology does not appear to work with tyrosyl phophorylation, and the methodology is additionally limited to a single application of phosphorylation.
In another approach, the peptide is proteolytically digested in 18O-enriched water whereby the carboxyl terminus of each peptide is isotopically labeled (Yao et al. (2001) Anal. Chem. 73: 2836-2842, U.S. Patent Publication No. 2002/0076817 to Figeys et al.). The isotopically labeled peptides are quantitated by comparison with a control sample digested in normal water where the mass spectrometer of the labeled peptide is shifted by 4 amu over the control. However, due to the small mass difference between the labeled peptide and the control, there are interferences with natural isotope distribution and resolution of the multiply charged peptides.
There remains a need, therefore, for compositions and methods for assaying and sequencing proteins and peptides.